Photoactivatable caged tamoxifen and tamoxifen derivative molecules and methods of use thereof

ABSTRACT

Provided herein are compositions containing photoactivatable caged tamoxifen and tamoxifen derivative molecules. Also provided are kits containing one of these compositions and a light source. Also provided are methods of optically inducing nuclear translocation of a fusion protein containing a mammalian estrogen receptor ligand binding domain in a eukaryotic cell and methods of optically inducing recombination in a eukaryotic cell that include contacting a eukaryotic cell with at least one of these compositions. Also provided are methods of treating breast cancer in a subject that include administering a photoactivatable caged tamoxifen or tamoxifen derivative molecule to a subject having breast cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.14/395,210, filed Oct. 17, 2014, which is a national phase filing under35 U.S.C. §371 of International application number PCT/US2013/031165,filed Mar. 14, 2013, which claims priority to U.S. Provisional PatentApplication Ser. No. 61/635,068, filed Apr. 18, 2012. The entirecontents of the parent applications are incorporated by referenceherein.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under grant number GrantNumbers CA141508, CA086355, CA092782, CA117969, HL070831, and SN268201awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Spatial and temporal genetic control is necessary to better dissect therole of specific genes and cell populations in development, disease, andtherapy. Site-specific recombination mediated by Cre recombinase is apowerful genetic tool to manipulate genetic elements in model organisms(Nagy, Genesis 26:99, 2000). When placed under the control of atissue-specific promoter, Cre/LoxP-mediated recombination allowstissue-specific investigation of gene functions (Nagy, Genesis 26:99,2000). Fusing Cre with a mutant form of the estrogen receptor (ER)ligand binding domain further enables temporal control of recombination(Nagy, Genesis 26:99, 2000). While this conditional CreER system allowsfor temporal control, the spatial control through tissue-specificpromoters is limited by relatively broad activation in all target cells,non-specificity of many of such cell type-‘specific’ promoters, and/orthe lack of validated promoters in certain cell types.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that conjugation oftamoxifen or tamoxifen derivatives to photoactivatable caged molecules(e.g., 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE)) results in asensitive and specific optical release of tamoxifen or tamoxifenderivatives to eukaryotic (e.g., mammalian, plant, yeast, nematode,parasite, or reptile) cells following irradiation. In view of thisdiscovery, provided herein are compositions containing photoactivatablecaged tamoxifen and caged tamoxifen derivative molecules, and methods ofoptically inducing nuclear translocation of a fusion protein containinga mammalian estrogen receptor ligand binding domain in a eukaryotic(e.g., mammalian, plant, yeast, nematode, parasite, or reptile) cell andmethods of optically inducing recombination in a eukaryotic (e.g.,mammalian, plant, yeast, nematode, parasite, or reptile) cell thatinclude contacting a eukaryotic cell with at least one of thesecompositions. Also provided are methods of treating breast cancer in asubject that include administering a photoactivatable caged tamoxifen orcaged tamoxifen derivative molecule to a subject having breast cancer.

Provided herein are compositions containing:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the composition further contains a physiologicallyacceptable excipient or buffer. In some embodiments, the composition isformulated as a liquid. In some embodiments, the composition isformulated for intravenous, intraarterial, transdermal, intramuscular,intraperitoneal, subcutaneous, intrathecal, interductal, periductal, orocular administration. In some embodiments, the composition isformulated as a solid. In some embodiments, the composition isformulated as a gel. In some embodiments, the composition is formulatedfor oral administration.

In some embodiments, the composition contains at least 80% by weight of

or a pharmaceutically acceptable salt thereof.

Also provided are kits containing one or more doses of any of thecompositions described herein; and a light source that emits light of awavelength of between 350 nm to 410 nm.

Also provided are methods of inducing nuclear translocation of a fusionprotein containing a human estrogen receptor ligand binding domain in aeukaryotic cell that include providing a eukaryotic cell that contains afusion protein containing a human estrogen receptor ligand bindingdomain, contacting the eukaryotic cell with any of the compositionsdescribed herein; and irradiating the eukaryotic cell with a wavelengthof light between 350 nm to 410 nm for a period of time sufficient torelease 4-hydroxycyclofen from the composition, where the released4-hydroxycyclofen induces the nuclear translocation of the fusionprotein. In some embodiments, the eukaryotic cell is in vitro. Someembodiments further include administering the irradiated eurkaryoticcell to a mammal. In some embodiments, the eukaryotic cell is in amammal (e.g., a mouse or rat). In some embodiments, the eukaryotic cellis present in the mammary gland or the skin. In some embodiments, thecomposition is locally administered to a target tissue in the mammalthat contains the eukaryotic cell. In some embodiments, the compositionis administered to the mammal via oral, intravenous, intraarterial,transdermal, intramuscular, intraperitoneal, subcutaneous, intrathecal,interductal, periductal, nasal, or ocular administration.

In some embodiments, the eukaryotic cell contains a nucleic acidencoding the fusion protein, and the nucleic acid is stably integratedinto a chromosome of the cell. In some embodiments, the nucleic acidencoding the fusion protein is operably linked to a tissue-specificpromoter sequence. In some embodiments, the nucleic acid encoding thefusion protein is operably linked to a ubiquitous promoter. In someembodiments, the nucleic acid encoding the fusion protein is operablylinked to an inducible promoter sequence, and the eukaryotic cell isfurther contacted with a chemical inducing agent.

In some embodiments, the fusion protein contains a sequence of arecombinase, and the fusion protein has recombinase enzymatic activity.In some embodiments, the recombinase is Cre recombinase. In someembodiments, the fusion protein contains a sequence of a transcriptionfactor, and the fusion protein is capable of promoting genetranscription in the nucleus of the eukaryotic cell. In someembodiments, the fusion protein promotes gene transcription of a genethat is present in a recombinant gene construct that is integrated intoa chromosome of the eukaryotic cell. In some embodiments, the fusionprotein contains a sequence of a transcription repressor, and the fusionprotein is capable of repressing transcription of a gene in the nucleusof the eurkaryotic cell. In some embodiments, the fusion proteinrepresses the transcription of a gene that is present in a recombinantgene construct that is integrated into a chromosome of the eukaryoticcell.

In some embodiments, the fusion protein contains a sequence of a histonedeacetylase, a histone acetyltransferase, or an O-6-methylguanine-DNAmethyltransferase, and the fusion protein has histone deacetylase,histone acetyltransferase, or O-6-methylguanine-DNA methyltransferaseactivity, respectively. In some embodiments, the fusion protein containsa sequence of a telomerase, and the fusion protein has telomeraseactivity. In some embodimennts, the fusion protein contains a sequenceof an oncogene.

In some embodiments, the eukaryotic cell is an epithelial cell, anendothelial cell, a muscle cell, an adipose cell, a bone cell, acartilage cell, or a neuron. In some embodiments, the eukaryotic cell isan undifferentiated cell, and the fusion protein comprises a sequence ofa transcription factor or transcription repressor that induces cellulardifferentiation. In some embodiments, the irradiating is performed forless than a total of 15 minutes. In some embodiments, the contacting andirradiating occur within 24 hours of each other. In some embodiments,the contacting and irradiating occur within 6 hours of each other. Insome embodiments, the contacting and irradiating occur within 1 hour ofeach other.

Also provided are methods of inducing recombination in a eukaryotic cellthat include: providing a eukaryotic cell that contains (i) a nucleicacid encoding a fusion protein containing a sequence of a recombinaseand a sequence of a human estrogen receptor ligand binding domain, wherethe fusion protein has recombinase activity, and (ii) a recombinaserecognition sequence that is specifically recognized by the fusionprotein, where both the nucleic acid encoding the fusion protein and therecombinase recognition sequence are integrated into a chromosome withinthe nucleus of the eukaryotic cell; contacting the eukaryotic cell anyof the compositions described herein; and irradiating the eukaryoticcell with a wavelength of light between 350 nm to 410 nm for a period oftime sufficient to release 4-hydroxycyclofen from the composition, wherethe 4-hydroxycyclofen stimulates the nuclear importation of the fusionprotein and the fusion protein stimulates recombination at therecombinase recognition sequence. In some embodiments, the eukaryoticcell is in vitro. In some embodiments, the eukaryotic cell is in amammal. In some embodiments, the mammal is a mouse or rat. In someembodiments, the eukaryotic cell is present in the mammary gland or theskin. In some embodiments, the composition is locally administered to atarget tissue in the mammal that contains the eukaryotic cell. In someembodiments, the composition is administered to the mammal via oral,intravenous, intraarterial, transdermal, intramuscular, intraperitoneal,subcutaneous, intrathecal, interductal, periductal, nasal, or ocularadministration.

In some embodiments, the recombinase is Cre recombinase. In someembodiments, the recombination results in a decrease in the expressionof a transgene located between two recombinase recognition sequences inthe chromosome. In some embodiments, the recombination results in thereplacement of a sequence between two recombinase recognition sequenceswith a new transgenic sequence. In some embodiments, the recombinationresults in the increase in the proximity of a promoter or enhancersequence to a transgene, wherein the recombination results in increasedexpression of the transgene.

In some embodiments, the nucleic acid encoding the fusion protein isoperably linked to tissue specific promoter sequence in the chromosomeof the eukaryotic cell. In some embodiments, the nucleic acid encodingthe fusion protein is operably linked to an inducible promoter in thechromosome of the eukaryotic cell, and the eukaryotic cell is furthercontacted with a chemical inducing agent. In some embodiments, thenucleic acid encoding the fusion protein is operably linked to aubiquitous promoter in the chromosome of the eukaryotic cell.

In some embodiments, the composition is administered to the animal bysystemic administration and a specific tissue of the mammal isirradiated. In some embodiments, the contacting and irradiating occurwithin 24 hours of each other. In some embodiments, the contacting andirradiating occur within 6 hours of each other. In some embodiments, thecontacting and irradiating occur within 1 hour of each other. In someembodiments, the irradiating is performed using an endoscopic lightsource.

Also provided are compositions that contain:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the composition further comprises at least oneadditional breast cancer therapeutic. In some embodiments, thecomposition further contains a physiologically acceptable excipient orbuffer. In some embodiments, the composition is formulated as a liquid.In some embodiments, the composition is formulated for intravenous,intraarterial, transdermal, intramuscular, intraperitoneal,subcutaneous, intrathecal, interductal, periductal, or ocularadministration. In some embodiments, the composition is formulated as asolid. In some embodiments, the composition is formulated as a gel. Insome embodiments, the composition is formulated for oral administration.

Also provided are methods of treating breast cancer in a subject thatinclude: administering to the subject an amount of any of thecompositions described herein sufficient to treat breast cancer in asubject; and irradiating the mammary tissue of the subject with lightbetween 200 nm and 900 nm, wherein the irradiating mediates the releaseof 4-hydroxytamoxifen in the irradiated mammary tissue. In someembodiments, the subject is diagnosed as having breast cancer. In someembodiments, the composition is locally administered to the mammarytissue of the subject. In some embodiments, the composition issystemically administered to the subject. In some embodiments, thecomposition is administered to the mammal via oral, intravenous,intraarterial, transdermal, intramuscular, intraperitoneal,subcutaneous, intrathecal, interductal, periductal, nasal, or ocularadministration. In some embodiments, the contacting and irradiatingoccur within 24 hours of each other. In some embodiments, the contactingand irradiating occur within 6 hours of each other. In some embodiments,the contacting and irradiating occur within 1 hour of each other. Someembodiments further include administering to the subject one or moreadditional breast cancer therapeutics.

Also provided are kits containing one or more doses of any of thecompositions described herein; and a light source that emits light of awavelength of between 200 nm to 900 nm.

By the term “mammalian estrogen receptor ligand binding domain” is meanta contiguous sequence of amino acids that is at least 80% (e.g., atleast 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to thesequence of the ligand binding domain of a mammalian (e.g., human,mouse, or rat) estrogen receptor that has the ability to bind totamoxifen or a tamoxifen derivative (e.g., 4-hydroxycyclofen).Non-limiting exemplary mammalian receptor ligand binding domains aredescribed herein. Methods for determining the ability of a mammalianestrogen receptor ligand binding domain to bind tamoxifen or a tamoxifenderivative are described herein.

By the term “human estrogen receptor ligand binding domain” is meant acontiguous sequence of amino acids that is at least 80% (e.g., at least85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence ofthe ligand binding domain of a human estrogen receptor that has theability to bind to tamoxifen or a tamoxifen derivative (e.g.,4-hydroxycyclofen). Non-limiting examples of human estrogen receptorligand binding domains are described herein. Additional examples ofhuman estrogen receptor ligand binding domains are known in the art. Insome embodiments, the human estrogen receptor ligand binding domaincontains a sequence at least 80% (e.g., at least 85%, 90%, 96%, 97%,98%, 99%, or 100%) identical to amino acids 310 to 547 of SEQ ID NO: 1.Methods for determining the ability of a human estrogen receptor ligandbinding domain to bind tamoxifen or a tamoxifen derivative are describedherein.

By the term “estrogen receptor fusion protein,” “ER fusion protein,” or“fusion protein containing an ER ligand binding domain” is meant apolypeptide sequence containing a mammalian estrogen receptor ligandbinding domain (e.g., a human estrogen receptor ligand binding domain)and at least one additional polypeptide sequence (a fusion partnerpolypeptide). Non-limiting examples of fusion partner polypeptides aredescribed herein. Additional examples of fusion partner polypeptides areknown in the art. In some embodiments, the fusion partner polypeptidecan be derived from a mammal (e.g., the same or a different mammal), abacterium, a virus, or a parasite. In some embodiments, the mammalianestrogen receptor ligand binding domain can be located at the N-terminusof the ER fusion protein or can be located N-terminal to the fusionpartner polypeptide. In some embodiments, the mammalian estrogenreceptor ligand binding domain can be located at the C-terminus of theER fusion protein or can be located C-terminal to the fusion partnerpolypeptide. In some embodiments, there can be an intervening amino acidsequence between the mammalian estrogen receptor ligand binding domainand the fusion partner polypeptide.

By the term “caged tamoxifen molecule” is meant a molecule containingtamoxifen conjugated to a photoactivatable caging molecule (e.g.,1-(4,5-dimethoxy-2-nitrophenyl) ethanol (DMNPE)) that releases tamoxifenupon exposure to light. Additional examples of photoactivatable cagingmolecules that can be present in caged tamoxifen molecules are describedherein. In some embodiments, there is a linking group present betweenthe tamoxifen and the photoactivatable caging molecule. In someembodiments, the tamoxifen and the photoactivatable caging molecule arejoined by an ester, an ether, an amido group, a carbonate, a carbamate,or an acetal.

By the term “caged tamoxifen derivative molecule” is meant a moleculecontaining tamoxifen derivative (e.g., 4-hydroxy tamoxifen or4-hydroxycyclofen) conjugated to a photoactivatable caging molecule(e.g., 1-(4,5-dimethoxy-2-nitrophenyl) ethanol (DMNPE)) that releasesthe tamoxifen derivative upon exposure to light. Additional examples ofphotoactivatable caging molecules that can be present in caged tamoxifenderivative molecules are described herein. In some embodiments, there isa linking group present between the tamoxifen derivative and thephotoactivatable caging molecule. In some embodiments, the tamoxifenderivative and the photoactivatable caging molecule are joined by anester, an ether, an amido group, a carbonate, a carbamate, or an acetal.

By the term “tissue-specific promoter sequence” is meant a nucleic acidsequence that is bound by a transcription factor and promotes thetranscription of an operably linked gene within a specific tissue in amammal. Non-limiting examples of tissue-specific promoter sequences aredescribed herein. Additional examples of tissue-specific promotersequences are known in the art.

By the term “inducible promoter sequence” is meant a nucleic acidsequence that is bound by a transcription factor and promotes thetranscription of an operably linked gene when the transcription factoris contacted with a chemical inducing agent (e.g., tetracycline).Non-limiting examples of inducible promoters and chemical inducingagents are described herein. Additional examples of inducible promotersand chemical inducing agents are known in the art.

By the term “ubiquitous promoter” is meant a nucleic acid sequence thatis bound by a transcription factor and promotes transcription of anoperably linked gene in a majority of the tissues present within amammal (e.g., β-actin promoter). Non-limiting examples of ubiquitouspromoters are described herein. Additional examples of ubiquitouspromoters are known in the art.

By the term “recombinase” is meant an enzyme that recognizes arecombinase recognition sequence present within a double stranded DNA,cleaves the double stranded DNA within or near the recombinaserecognition sequence, and ligates at least one of the severed ends ofthe double-stranded DNA to form a contiguous piece of DNA. In someembodiments, the activity of a recombinase results in the deletion of anucleic acid sequence located between two different recombinaserecognition sequences. In some embodiments, the activity of arecombinase results in the replacement of the sequence between twodifferent recombinase recognition sequences with a different nucleicacid sequence (a different nucleic acid sequence that has a recombinaserecognition sequence located at its 5′ and 3′ ends). Non-limitingexamples of recombinases and recombinase recognition sequences aredescribed herein. Additional examples of recombinases and recombinaserecognition sequences are known in the art.

By the term “recombinant gene construct” is meant a heterologousartificial gene construct that is stably introduced into a chromosome ofa eukaryotic (e.g., mammalian, plant, yeast, nematode, parasite, orreptile) cell. In some embodiments, the recombinant gene constructcontains a sequence encoding an ER fusion protein (e.g., any of the ERfusion proteins described herein). In some embodiments, the recombinantgene construct contains a sequence encoding an ER fusion proteinoperatively linked to one or more promoter or enhancer sequences (e.g.,an inducible, tissue-specific, or ubiquitous promoter). In someembodiments, the recombinant gene construct contains a nucleic acidsequence that contains at least one recombinase recognition sequence(e.g., any of the recombinase recognition sequences described herein orknown in the art).

As is used throughout, the term “transgenic” refers to a eukaryotic(e.g., mammalian, plant, yeast, nematode, parasite, or reptile) cell invitro or a non-human eukaryote (e.g., mammal) that contains at least onerecombinant gene construct.

By the term “transcription repressor” is meant a protein that binds to apromoter sequence in DNA and mediates a decrease in the transcription ofa nucleic acid sequence operatively linked to the promoter sequence.

By the term “transcription factor” or “transcription activator” is meanta protein that binds to a promoter sequence in DNA and mediates anincrease in the transcription of a nucleic acid sequence operativelylinked to the promoter sequence.

By the term “oncogene” is meant a polypeptide that contributes to thedevelopment of cancer in a mammal. In some embodiments, the oncogeneresults in a decreased function in an apoptotic signaling pathway in amammalian cell. In some embodiments, the oncogene results in thederegulation of the cell cycle in a mammalian cell. A variety ofdifferent oncogenes are known in the art.

Other definitions appear in context throughout this disclosure. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Methods and materials are describedherein for use in the present invention; other, suitable methods andmaterials known in the art can also be used. The materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, sequences, database entries,and other references mentioned herein are incorporated by reference intheir entirety. In case of conflict, the present specification,including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the photoactivation-dependent CreER/loxPsystem in the Rosa26CreER^(T2); mT/mG reporter mice.

FIG. 2 is a schematic showing the synthesis of caged 4-OHC usingMitsonobu reaction conditions.

FIG. 3 is a schematic showing the photocleavage reaction of caged 4-OHCusing 365 nm UV light.

FIG. 4 is a set of absorbance spectra that show the changes in UV-Visabsorbance that occur during the photocleavage of caged 4-OHC inwater:acetonitrile (1:1 v/v).

FIG. 5 is a graph showing the change in absorbance of caged 4-OHC at 375nm over UV irradiation time.

FIG. 6 is a set of three HPLC-MS chromatograms showing the quantitativerelease of 4-OHC from caged 4-OHC upon UV irradiation. The threechromatograms represent a UV exposure of 0 minute, 1 minute, and 10minutes. The peaks corresponding to caged 4-OHC (*) and released 4-OHC(**) are indicated.

FIG. 7 is a set of eight fluorescent micrographs of mouse embryonicfibroblasts (MEFs) isolated from Rosa26CreER^(T2); mT/mG mice followingtreatment with vehicle alone (two left panels), 4-OHC (two left centerpanels), caged 4-OHC (two right center panels), and caged 4-OHC withlight exposure (two right panels).

FIG. 8 is a graph showing the percentage of enhanced green fluorescentprotein (EGFP)-positive MEFs (normalized to 4-OHC-treated MEF controls)in MEFs treated with 4-OHC or caged 4-OHC following different durationsof UV activation. The “intracellular+medium” group represents cellsamples without PBS wash before UV irradiation. The “intracellular only”group represents cell samples washed twice with PBS before UVirradiation.

FIG. 9 is a schematic of the photoactivation-dependent CreER/loxP systemin the Rosa26CreER^(T2);R26R reporter mice. Photoactivated release oflacZ gene expression occurs in illuminated and caged 4-OHC-treatedcells.

FIG. 10 is a set of four micrographs of X-gal stained MEFs fromRosa26CreER^(T2);R26R mice embryos following treatment with vehiclealone (upper left panel), 4-OHC (upper right panel), caged 4-OHC (bottomleft panel), and caged 4-OHC with 365 nm light exposure (bottom rightpanel).

FIG. 11 is a set of four photomicrographs showing the EGFP expressioninduced by 365 nm UV activation of caged 4-OHC in mammary epithelialcells isolated from Rosa26CreER^(T2); mT/mG female mice and cultured toform acini on Matrigel. The images shown are projections of the Z-stackconfocal images.

FIG. 12 is a graphic showing the culturing of mammary epithelial cellacini from Rosa26CreER^(T2); mT/mG female mice and two fluorescentmicrographs of individual acini. The acini were illuminated with the 60×objective of an inverted epifluorescent microscope equipped with astandard DAPI filter set for 1 minute after treatment with 5 μM caged4-OHC and brief PBS wash. The fluorescent micrographs were collectedfour days later from the live acini using a laser scanning confocalmicroscope. The left panel is a micrograph of an acini treated withcaged 4-OHC and exposed to light, and the right panel is a micrograph ofan acini treated with caged 4-OHC and not exposed to light.

FIG. 13 is a set of six images showing the expression of EGFP inRosa26CreER^(T2); mT/mG mice following treatment with 4-OHC. EGFPexpression was detected using an OV-110 epifluorescent imager. Micetreated with vehicle control and irradiated with 365 nm UV light areshown in the left panels. Mice treated with 4-OHC are shown in the rightpanels.

FIG. 14 is a set of two images showing the EGFP expression in theventral skin of Rosa26CreER^(T2); mT/mG reporter mice followingtreatment with caged 4-OHC and no light treatment (left panel) or caged4-OHC with 365 nm light treatment. An increase in EGFP signal isfollowed by a decrease in tdTomato signal; low values of EGFP/tdTomatoratio correspond to low EGFP and high tdTomato, while high values ofratios correspond to high EGFP and low tdTomato.

FIG. 15 is a schematic of the experimental design for intraperitonealinjection of vehicle or caged 4-OHC into the Rosa26CreER^(T2); mT/mGfemale mice and subsequent 365 nm illumination on the right inguinal(#4) mammary fat pad.

FIG. 16 is a set of four images of ex vivo mammary tissue fromRosa26CreER^(T2); mT/mG female mice intraperitoneally injected withvehicle and exposed (right panels) or unexposed (left panels) to 365 nmlight. The mammary tissue was imaged with an IV-110 epifluorescentimager.

FIG. 17 is a set of four images of ex vivo mammary tissue fromRosa26CreER^(T2); mT/mG female mice intraperitoneally injected withcaged 4-OHC and exposed (right panels) or unexposed (left panels) to 365nm light. The mammary tissue was imaged with an IV-110 epifluorescentimager. The illuminated mammary gland tissues injected with caged 4-OHCexpressed EGFP.

FIG. 18 is a set of eight photomicrographs showing the X-gal stainedmouse embryonic fibroblasts from Rosa26-CreERT2; Lox-STOP-Lox-lacZembryos following no treatment (upper left panel), treatment with 5 μM4-OHC (positive control; lower left panel), treatment with CagedMolecule I (“Compound I”) with or without 365 nm UV light exposure (topright and top center panels, respectively), treatment with CagedMolecule II (“Compound II”) with or without 365 nm UV light exposure(middle right and middle center panels, respectively), or treatment withCaged Molecule III (“Compound III”) with or without 365 UV lightexposure (bottom right and bottom left panels, respectively).

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, on the discovery that photoactivatablecaged tamoxifen and caged tamoxifen derivative molecules can be used toselectively and sensitively deliver tamoxifen or tamoxifen derivativesto cells following irradiation. Thus, provided herein are compositionscontaining these caged tamoxifen and caged tamoxifen derivativemolecules, and methods of inducing nuclear translocation of an ER fusionprotein in a eukaryotic (e.g., mammalian, plant, yeast, nematode,parasite, or reptile) cell and methods of inducing recombination in aeukaryotic (e.g., mammalian, plant, yeast, nematode, parasite, orreptile) cell that include contacting a eukaryotic cell with one of thecaged tamoxifen or caged tamoxifen derivative molecules and irradiatingthe eukaryotic cell. Also provided are methods of treating breast cancerin a subject that include administering a photoactivatable cagedtamoxifen or caged tamoxifen derivative molecule to a subject havingbreast cancer. Various, non-limiting features of each aspect of theinvention are described below.

Compounds

Provided herein are caged tamoxifen and caged tamoxifen derivativemolecules that are sensitive to light-induced degradation. Lightexposure of the caged tamoxifen or caged tamoxifen derivative moleculestriggers the release of the tamoxifen or tamoxifen derivative. Any ofthe caged tamoxifen or caged tamoxifen derivative molecules describedherein can be used in any of the methods described herein.

A prior version of caged tamoxifen (4-hydrocyclofen conjugated with thephotoactivatable 4,5-demethoxy-2-nitrophenl methanol (DMNPM) groupdemonstrated spontaneous breakdown in the absence of appropriate lightexposure. These results indicated that the DMNPM caging group or theDMNPM caged 4-hydroxycyclofen can be unstable in the absence ofappropriate light exposure. In addition, the photocleavage of the DMNPMcaged 4-hydroxycyclofen results in the generation of a reactive aldehydegroup (shown in Schematic #1 below). This reactive aldehyde product maytrigger unwanted side reactions with amine containing biomolecules inthe cell. This process can lead to unwanted, detrimental chemical sidereactions in the cell.

Provided herein are caged tamoxifen or caged tamoxifen derivativemolecules that have improved stability in the absence of appropriatelight exposure. In some embodiments, the caged tamoxifen or cagedtamoxifen derivative molecules contain tamoxifen or a tamoxifenderivative (e.g., 4-hydroxycyclofen) conjugated to1-(4,5-dimethoxy-2-nitrophenyl) ethanol (DMNPE). In some embodiments,the caged tamoxifen or caged tamoxifen derivative is conjugated tophotoactivatable caging group selected from the group of:carboxy-2-nitrobenzyl (CNB); 4,5-dimethoxy-2-nitrobenzyl (DMNB);2,2′-dinitrobenzhydryl (DNBH); 1-(2-nitrophenyl)ethyl (NPE);(4,4′-bis-{8-[4-nitro-3-(2-propyl)-styryl]}-3,3′-dimethoxybiphenyl(BNSMB);(2,7-bis-{4-nitro-8-[3-(2-propyl)-styryl]}-9,9-bis-[1-(3,6-dioxaheptyl)]-fluorene(BNSF); 4-carboxymethoxy-5,7-dinitroindolinyl (CDNI); 2-nitrobenzyl and7-nitroindoline derivatives; coumarin-4-ylmethyl;8-bromo-7-hydroxyquinoline derivatives; p-hydroxyphenacyl; and heavymetal. In some embodiments, the photoactivatable caging molecule isconnected to the tamoxifen or tamoxifen derivative by an ester, anether, an amido group, a carbonate, a carbamate, or an acetal.

In some embodiments, the tamoxifen derivative contains a modification tothe triphenyl ethylene skeleton. Non-limiting examples of tamoxifenderivatives include 4-hydroxycyclofen and 4-hydroxytamoxifen. Additionalnon-limiting examples of tamoxifen derivatives are shown below(Schematic #2).

In some embodiments, the caged tamoxifen derivative molecule is1-(4,5-dimethoxy-2-nitrophenyl) ethanol (DMNPE) caged 4-hydroxycyclofen(DMNPE-4-OHC). DMNPE-4-OHC did not appear to show any sign of breakdownprior to illumination (essentially inert in the dark). DMNPE-4-OHC canbe decaged using a wavelength of light between 350 nm and 405 nm (seeExample). The light-induced breakdown of DMNPE-4-OHC generates a ketoneproduct (shown in Schematic #3 below), rather than a reactivealdehyde-containing product. The production of a ketone product reducesthe possibility of any detrimental side reactions within a cell.

In some embodiments, the caged tamoxifen derivative is one of thestructures shown in Schematic #4 below.

In some embodiments, the caged tamoxifen derivative is one of the threestructures shown in Schematic #5 below.

Caged Molecules I, II, and III (shown in Schematic #5) were tested incell culture experiments and were able to induce nuclear translocationof an ER fusion protein in vitro (see, FIG. 17). Caged Molecule III(shown in Schematic #5) is a two-photon activatable caged tamoxifenderivative. Activation using a two-photon light source is particularlybeneficial for in vivo study due to its ability to allow deep tissuepenetration and low tissue damaging effect. A two-photon light sourceemits wavelengths that are greater than 600 nm (e.g., emits a wavelengththat is between 700 nm and 800 nm, between 700 nm and 900 nm, between650 nm and 850 nm, between 500 nm and 800 nm, or between 600 nm and 900nm). In some embodiments where the nuclear translocation of an ER fusionprotein within an internal organ is desired, a mammal is administered acomposition containing Caged Molecule III (shown in Schematic #5 above).

In some embodiments, the caged tamoxifen derivative is DMNPE-conjugated4-hydroxy tamoxifen (shown in Schematic #6 below). 4-hydroxy tamoxifenis the active metabolite form of tamoxifen. As described below, theDMNPE-conjugated 4-hydroxy tamoxifen can be used as a prodrug forlight-induced localized treatment of breast cancer in a subject.

Synthetic methods for conjugating tamoxifen or a tamoxifen derivative(e.g., any of the tamoxifen derivatives described herein) to aphotoactivatable caging molecule (e.g., any of the caging moleculesdescribed herein) are known in the art. For example, DMNPE can beconjugated to a tamoxifen derivative (e.g., 4-hydroxycyclofen) usingMitsonobu coupling conditions (see, e.g., the method described in theExample). In some embodiments, the tamoxifen or tamoxifen derivative canbe conjugated to a bromo derivative of the photoactivatable-cagedmolecule (e.g., DMNPE) via a nucleaophilic substitution reaction. Afterthe synthesis, the caged tamoxifen or caged tamoxifen derivativemolecule can be purified, e.g., using a SiO₂ (see, e.g., the methoddescribed in the Example). In some embodiments, the caged tamoxifen orcaged tamoxifen derivative molecule can be at least 80% (e.g., at least85%, 90%, 95%, or 99%) pure by weight.

Compositions and Kits

Provided herein are compositions containing at least one of any of thecaged tamoxifen molecules or caged tamoxifen derivative moleculesdescribed herein. In some embodiments, the composition contains at least80% (e.g., at least 85%, 90%, 95%, or 99%) of the caged tamoxifen or thecaged tamoxifen derivative molecule by weight. In some embodiments, thecomposition contains a pharmaceutically acceptable excipient or buffer(e.g., saline, DMSO, PEG400, an acetate buffer, VitE-TPGS, ethanol,Solutol, cremophor, Tween, and mixtures thereof). In some embodiments,the compositions are formulated using a combination of ethanol, apolyethylene glycol, Tween, and Solutol. In some embodiments, thecompositions are formulated in 10% N,N-dimethylacetamide (DMAC) and 10%Solutol in saline.

In some embodiments, the compositions are formulated as a liquid forsystemic administration. In some embodiments, the compositions areformulated for intraarterial, intravenous, intraperitoneal, intrathecal,ocular, nasal, intramuscular, intraductal, or subcutaneousadministration.

In some embodiments, the compositions are formulated as a solid. In someembodiments, the compositions are formulated for oral or topical (e.g.,transdermal) administration. In some embodiments, the compositions areformulated as a suppository.

In some embodiments, the compositions are encapsulated in nanomaterialsfor targeted delivery (e.g., encapsulated in a nanomaterial having oneor more tissue- or cell-targeting molecules on its surface). In someembodiments, the compositions are formulated as an emulsion or as aliposome-containing composition. In some embodiments, the compositionscan contain dimers, oligomers, or polymers of any of the caged tamoxifenor caged tamoxifen derivative molecules described herein. In someembodiments, the caged tamoxifen or caged tamoxifen derivative moleculesare formulated for sustained release (e.g., formulated in abiodegradable polymers or in nanoparticles). In some embodiments, thecompositions are formulated in an implantable device that allows forsustained release of the caged tamoxifen or caged tamoxifen derivativemolecules.

Pharmaceutical compositions are formulated to be compatible with theirintended route of administration or the intended target tissue or cell,e.g., systemic or local administration. In some embodiments, thecompositions are formulated for oral, intravenous, intradermal,subcutaneous, transmucosal (e.g., nasal sprays are formulated forinhalation), or transdermal (e.g., topical ointments, salves, gels,patches, or creams as generally known in the art) administration. Thecompositions can include a sterile diluent (e.g., sterile water orsaline), a fixed oil, polyethylene glycol, glycerine, propylene glycol,or other synthetic solvents; antibacterial or antifungal agents, such asbenzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like; antioxidants, such as ascorbic acid or sodiumbisulfate; chelating agents, such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates, or phosphates; and isotonic agents,such as sugars (e.g., dextrose), polyalcohols (e.g., manitol orsorbitol), or salts (e.g., sodium chloride). Liposomal suspensions canalso be used as pharmaceutically acceptable carriers (see, e.g., U.S.Pat. No. 4,522,811; herein incorporated by reference). Preparations ofthe compositions can be formulated and enclosed in ampules, disposablesyringes, or multiple dose vials that prevent exposure of the cagedtamoxifen or caged tamoxifen derivative molecules to light. Whererequired (as in, for example, injectable formulations), proper fluiditycan be maintained by, for example, the use of a coating such aslecithin, or a surfactant. Absorption of the caged tamoxifen or cagedtamoxifen derivative molecules can be prolonged by including an agentthat delays absorption (e.g., aluminum monostearate and gelatin).Alternatively, controlled release can be achieved by implants andmicroencapsulated delivery systems, which can include biodegradable,biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid; AlzaCorporation and Nova Pharmaceutical, Inc.).

Where oral administration is intended, the agent can be included inpills, capsules, troches and the like, and can contain any of thefollowing ingredients, or compounds of a similar nature: a binder, suchas microcrystalline cellulose, gum tragacanth, or gelatin; an excipient,such as starch or lactose; a disintegrating agent, such as alginic acid,Primogel, or corn starch; a lubricant, such as magnesium stearate; aglidant, such as colloidal silicon dioxide; a sweetening agent, such assucrose or saccharin; or a flavoring agent, such as peppermint, methylsalicylate, or orange flavoring.

The compositions described herein can be formulated for ocular orparenteral (e.g., oral) administration in dosage unit form (i.e.,physically discrete units containing a predetermined quantity of activecompound for ease of administration and uniformity of dosage). Toxicityand therapeutic efficacy of compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals. Onecan, for example, determine the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population), the therapeutic index being the ratio of LD50:ED50.Agents that exhibit high therapeutic indices are preferred. Where anagent exhibits an undesirable side effect, care should be taken totarget that agent to the site of the affected or targeted tissue (theaim being to minimize potential damage to unaffected cells and, thereby,reduce side effects). Toxicity and therapeutic efficacy can bedetermined by other standard pharmaceutical procedures.

In some embodiments, the composition containing a caged tamoxifen orcaged tamoxifen derivative molecule is formulated in a single dosageform (e.g., a single dosage form containing between 1 mg to 500 mg,between 1 mg and 400 mg, between 1 mg and 300 mg, between 1 mg and 250mg, between 1 mg and 200 mg, between 1 mg and 100 mg, and between 1 mgand 50 mg of the caged tamoxifen or caged tamoxifen derivativemolecule).

In some embodiments, the compositions contain at least one cagedtamoxifen molecule or at least one caged tamoxifen derivative moleculeand one or more additional breast cancer therapeutics (e.g., any of thebreast cancer therapeutics described herein).

Also provided herein are kits that contain at least one dose of any ofthe compositions described herein. In some embodiments, the kits canfurther include an item for use in administering a composition (e.g.,any of the compositions described herein) to the subject (e.g., asyringe, e.g., a pre-filled syringe). In some embodiments, the kitscontain one or more doses (e.g., at least two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, twenty,thirty, or forty doses) (e.g., oral doses) of any of the compositionsdescribed herein. In some embodiments, the kit further containsinstructions for administering the composition (or a dose of thecomposition) to a mammal (e.g., a non-human transgenic mammal or a humanhaving breast cancer). In some embodiments, the kits contain acomposition containing at least one of the caged tamoxifen molecules orat least one of the caged tamoxifen derivative molecules, and acomposition containing at least one additional breast cancer therapeutic(e.g., any of the additional breast cancer therapeutics describedherein). In some embodiments, the kit further contains instructions forperforming any of the methods described herein

In some embodiments, the kits can further contain a light source (e.g.,any of the light sources described herein, e.g., a hand-held lightsource or a light source that can be used in an endoscopic ororthroscopic procedure). In some embodiments, the kits contain atwo-photon light source (e.g., a hand-held two photon light source). Insome embodiments, a light source emits a wavelength of light that isbetween 700 nm and 800 nm, between 700 nm and 900 nm, between 650 nm and850 nm, between 500 nm and 800 nm, or between 600 nm and 900 nm. In someembodiments, the light source emits light at a wavelength ofapproximately 730 nm.

Light Sources

Light sources that emit light between 200 nm and 900 nm (e.g., between300 nm and 700 nm, between 300 nm and 600 nm, between 300 nm and 500 nm,between 350 nm and 410 nm, between 200 nm and 700 nm, and between 700 nmand 900 nm) that can be used to breakdown the caged tamoxifen or cagedtamoxifen derivative molecules (e.g., any of the caged tamoxifen orcaged tamoxifen derivative molecules described herein). In someembodiments, the light source emits a wavelength of between 700 nm and800 nm, between 700 nm and 900 nm, between 650 nm and 850 nm, between500 nm and 800 nm, or between 600 nm and 900 nm. In some embodiments, alight source that emits light of 350 nm to 410 nm is used to breakdownDMNPE-caged 4-hydroxycyclofen.

In some embodiments, the light source can be directed to a particulartissue or cell that is expressing the ER fusion protein (e.g., any ofthe ER fusion proteins described herein) and that has been contactedwith any of the caged tamoxifen or caged tamoxifen derivative moleculesdescribed herein. In some embodiments, the light source is used toirradiate an animal containing a cell containing the ER fusion proteinthat has been contacted with any of the caged tamoxifen or cagedtamoxifen derivative molecules described herein. In some embodiments,the light source is a hand-held device. In some embodiments, the lightsource is placed at the end of an endoscope or orthoscopic device. Insome embodiments, the light source is inserted into a tissue in the bodythrough the use of a catheter. In some embodiments, the light source isa two-photon light source.

ER Fusion Proteins

Estrogen receptor (ER)-fusion proteins as described herein are proteinsthat contain a mammalian estrogen receptor ligand binding domain and anadditional polypeptide sequence (a fusion partner polypeptide). In someembodiments, the ER-fusion protein contains a human estrogen receptorligand binding domain. In some embodiments, the mammalian (e.g., human)estrogen receptor ligand binding domain is located at the N-terminus ofthe ER-fusion protein or is located N-terminal to the additionalpolypeptide sequence (fusion partner polypeptide). In some embodiments,the mammalian (e.g., human) estrogen receptor ligand binding domain islocated at the C-terminus of the ER fusion protein or is locatedC-terminal to the additional polypeptide sequence (fusion partnerpolypeptide).

Mammalian Estrogen Receptor Ligand Binding Domains

In some embodiments, the mammalian estrogen receptor ligand bindingdomain contains a contiguous sequence that is at least 80% (e.g., atleast 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to acontiguous sequence present in a human estrogen ligand binding domain,and has the ability to bind to tamoxifen or a tamoxifen derivative(e.g., any of the tamoxifen derivatives described herein). In someembodiments, the mammalian estrogen receptor ligand binding domaincontains a sequence that is at least 80% (e.g., at least 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence presentbetween amino acids 310 to 547 of SEQ ID NO: 1 (underlined below). Insome embodiments, amino acids 346, 351, 353, 387, 394, 521, 524 of SEQID NO: 1 are conserved (underlined and in bold below).

Human Estrogen Receptor Protein (SEQ ID NO: 1) 1mtmtlhtkas gmallhqiqg neleplnrpq lkiplerplg evyldsskpa vynypegaay 61efnaaaaana qvygqtglpy gpgseaaafg snglggfppl nsyspsplml lhpppqlspf 121lqphgqqvpy ylenepsgyt vreagppafy rpnsdnrrqg grerlastnd kgsmamesak 181etrycavcnd yasgyhygvw scegckaffk rsiqghndym cpatnqctid knrrkscqac 241rlrkcyevgm mkggirkdrr ggrmlkhkrq rddgegrgev gsagdmraan lwpsplmikr 301skknslalsl tadqmvsall daeppilyse ydptrpfsea smmglltnla drelvhminw 361akrvpgfvdl tlhdqvhlle cawleilmig lvwrsmehpg kllfapnlll drnqgkcveg 421mveifdmlla tssrfrmmnl qgeefvclks iillnsgvyt flsstlksle ekdhihrvld 481kitdtlihlm akagltlqqq hqrlaqllli lshirhmsnk gmehlysmkc knvvplydll 541lemldahrlh aptsrggasv eetdqshlat agstsshslq kyyitgeaeg fpatv

An exemplary cDNA encoding a human estrogen receptor protein is SEQ IDNO: 2.

Additional examples of human estrogen receptor ligand binding domainsthat can be used in the fusion proteins are described in Pfannkuche etal., Biotechniques 48:113-120, 2010; Andersson et al., Transgenic Res.19:715-725, 2010; Maeda et al., Bone 46:472-478, 2010; Tumurbaatar etal., J. Virol. Methods 146:5-13, 2007; Dwomiczak et al., Nephron Exp.Nephrol. 106:e11-e20, 2007; Weber et al., Biol. Reprod. 68:553-539,2003; Hayashi et al., Dev. Biol. 244:305-318, 2002; Vallier et al.,Proc. Natl. Acad. Sci. U.S.A. 98:2467-2472, 2001; Fuhrmann-Benzakein etal., Nucleic Acids Res. 28:E99, 2000; Indra et al., Nucleic Acids Res.27:4324-4327, 1999; and Metzger et al., Proc. Natl. Acad. Sci. U.S.A.92:6991-6995, 1995. These references describe Cre recombinase-humanestrogen receptor ligand binding domain fusion proteins. Sequencesencoding the human estrogen receptor ligand binding domain are describedin these references, and these sequences can be isolated and used toform any of the ER fusion proteins described herein using molecularbiology methods known in the art.

In some embodiments, the mammalian estrogen receptor ligand bindingdomain contains a contiguous sequence that is at least 80% (e.g., atleast 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to acontiguous sequence present in a mouse estrogen receptor ligand bindingdomain. In some embodiments, the mammalian estrogen receptor ligandbinding domain contains a sequence that is at least 80% (e.g., at least85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a contiguoussequence present between amino acids 314 to 551 of SEQ ID NO: 3(underlined below). In some embodiments, amino acids 350, 355, 357, 391,398, 525, and 528 of SEQ ID NO: 3 are conserved (underlined and in boldbelow).

Mouse Estrogen Receptor Protein (SEQ ID NO: 3) 1mtmtlhtkas gmallhqiqg nelepinrpq lkmpmeralg evyvdnskpt vfnypegaay 61efnaaaaaaa aasapvygqs giaygpgsea aafsanslga fpqlnsysps plmllhpppq 121lspflhphgq qvpyylenep sayavrdtgp pafyrsnsdn rrqngrerls ssnekgnmim 181esaketryca vcndyasgyh ygvwscegck affkrsiqgh ndymcpatnq ctidknrrks 241cqacrlrkcy evgmmkggir kdrrggrmlk hkrqrddleg rnemgasgdm raanlwpspl 301vikhtkknsp alsltadqmv salldaeppm iyseydpsrp fseasmmgll tnladrelvh 361minwakrvpg fgdlnlhdqv hllecawlei lmiglvwrsm ehpgkllfap nllldrnqgk 421cvegmveifd mllatssrfr mmnlqgeefv clksiillns gvytflsstl ksleekdhih 481rvldkitdtl ihlmakaglt lqqqhrrlaq lllilshirh msnkgmehly nmkcknvvpl 541ydlllemlda hrlhapasrm gvppeepsqt qlattsstsa hslqtyyipp eaegfpnti

An exemplary cDNA encoding a mouse estrogen receptor protein is SEQ IDNO: 4.

In some embodiments, the mammalian estrogen receptor ligand bindingdomain contains a contiguous sequence that is at least 80% (e.g., atleast 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to acontiguous sequence present in a rat estrogen receptor ligand bindingdomain. In some embodiments, the mammalian estrogen receptor ligandbinding domain contains a sequence that is at least 80% (e.g., at least85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a contiguoussequence present between amino acids 315 to 552 of SEQ ID NO: 5(underlined below). In some embodiments, amino acids 351, 356, 358, 392,399, 526, and 529 of SEQ ID NO: 5 are conserved (underlined and in boldbelow).

Rat Estrogen Receptor Protein (SEQ ID NO: 5) 1mtmtlhtkas gmallhqiqg nelepinrpq lkmpmeralg evyvdnskpa vfnypegaay 61efnaaaaaaa agasapvygq ssitygpgse aaafganslg afpqlnsvsp splmllhppp 121hvspflhphg hqvpyylene psayavrdtg ppafyrsnsd nrrqngrerl ssssekgnmi 181mesaketryc avcndyasgy hygvwscegc kaffkrsiqg hndymcpatn qctidknrrk 241scqacrlrkc yevgmmkggi rkdrrggrml khkrqrddle grnemgtsgd mraanlwpsp 301lvikhtkkns palsltadqm vsalldaepp liyseydpsr pfseasmmgl ltnladrelv 361hminwakrvp gfgdlnlhdq vhllecawle ilmiglvwrs mehpgkllfa pnllldrnqg 421kcvegmveif dmllatssrf rmmnlqgeef vclksiilln sgvytflsst lksleekdhi 481hrvldkindt lihlmakagl tlqqqhrrla qlllilshir hmsnkgmehl ynmkcknvvp 541lydlllemld ahrlhapasr mgvppeepsq sqltttssts ahslqtyyip peaegfpnti

An exemplary cDNA encoding a rat estrogen receptor protein is SEQ ID NO:6.

The ability of an ER fusion protein to bind to tamoxifen or a tamoxifenderivative can be assessed directly using high performance liquidchromatograph-mass spectrometry (HPLC-MS) or circular dichroism (see,e.g., Nair et al., J. Mol. Endocrinol. 35:211-223, 2005). The ability ofan ER fusion protein to bind tamoxifen or a tamoxifen derivative canalso be assessed indirectly by the detection of the nucleartranslocation of an ER fusion protein (e.g., detected byimmunofluorescent microscopy or induced expression of a reportertransgene integrated within a chromosome of the cell) or the detectionof an activity of the ER fusion protein within the nucleus of the cell.Exemplary methods for detecting tamoxifen- or tamoxifenderivative-induced nuclear translocation are described in the Examples.Methods for detecting the activity of an ER fusion protein within thenucleus of the cell depend on the specific activity of the ER fusionprotein. Methods to detect the activity of an ER fusion protein withinthe nucleus of the cell are also known in the art.

Fusion Partner Polypeptides

ER fusion proteins can contain a fusion partner polypeptide from anysource (e.g., a human, mouse, rat, bacterial, viral, or parasiticpolypeptide sequence). In some embodiments, the fusion partnerpolypeptide contains a polypeptide sequence of a recombinase (e.g., aCre recombinase from P1 phage, FLP from S. cerevisiae, λ, integrase fromlambda phage, gamma-delta resolvase from Tn1000 transposon, Tn3resolvase from the Tn3 transposon, and φC31 integrase from φC31 phage)and the fusion protein has recombinase activity in a cell treated withtamoxifen or a tamoxifen derivative. Non-limiting exemplary sequences ofCre recombinase polypeptides that can be included in the ER fusionproteins, as well as exemplary ER fusion proteins containing a Crerecombinase polypeptide are described in Pfannkuche et al.,Biotechniques 48:113-120, 2010; Andersson et al., Transgenic Res.19:715-725, 2010; Maeda et al., Bone 46:472-478, 2010; Tumurbaatar etal., J. Virol. Methods 146:5-13, 2007; Dwomiczak et al., Nephron Exp.Nephrol. 106:e11-e20, 2007; Weber et al., Biol. Reprod. 68:553-539,2003; Hayashi et al., Dev. Biol. 244:305-318, 2002; Vallier et al.,Proc. Natl. Acad. Sci. U.S.A. 98:2467-2472, 2001; Fuhrmann-Benzakein etal., Nucleic Acids Res. 28:E99, 2000; Indra et al., Nucleic Acids Res.27:4324-4327, 1999; and Metzger et al., Proc. Natl. Acad. Sci. U.S.A.92:6991-6995, 1995. Additional non-limiting polypeptide sequences offusion partner polypeptides are listed in Table 1 below. Methods fordetecting recombinase activity of a fusion protein in a cell treatedwith tamoxifen or a tamoxifen derivative are known in the art.Non-limiting exemplary methods for detecting recombinase activity of afusion protein in a cell treated with tamoxifen or a tamoxifenderivative are described in the Example.

TABLE 1 Exemplary Recombinase Fusion Partner Polypeptide Sequences CreRecombinase NCBI Accession No. AFA52013.1; nucleotides 2590 to 3621 ofNCBI Accession No. JN798465.1 Flp Recombinase NCBI Accession No.AAT08996.1; nucleotides 6054 to 7325 of NCBI Accession No. AY597273.1Lambda Integrase NCBI Accession No. ADW76527.1; nucleotides 108705 to109829 of NCBI Accession No. CP002506.1 Gamma Delta NCBI Accession No.P03012.1 Resolvase Tn3 Resolvase NCBI Accession No. AEZ43735.1;nucleotides 1829 to 2386 of NCBI Accession No. CP003112.1 φC31 IntegraseNCBI Accession No. CAI94541.1; nucleotides 5011 to 6852 of NCBIAccession No. AJ937361.1

In some embodiments, the fusion partner polypeptide contains apolypeptide sequence of a transcription factor (e.g., a transcriptionfactor that plays a role in cellular differentiation) and the fusionprotein is capable of inducing gene transcription in a cell treated withtamoxifen or a tamoxifen derivative. In some embodiments, the fusionpartner polypeptide contains a polypeptide sequence of a transcriptionrepressor (e.g., a transcription repressor that plays a role in cellulardifferentiation) and the fusion protein is capable of inhibiting genetranscription in a cell treated with tamoxifen or a tamoxifenderivative. In some embodiments, the fusion partner polypeptide containsa polypeptide sequence of a histone deacetylase and the fusion proteinhas histone deacetylase activity in a cell treated with tamoxifen or atamoxifen derivative. In some embodiments, the fusion partnerpolypeptide contains a polypeptide sequence of an O-6-methylguanine-DNAmethyltransferase and the fusion protein has O-6-methylguanine-DNAmethyltransferase activity in a cell treated with tamoxifen or atamoxifen derivative. In some embodiments, the fusion partnerpolypeptide contains a polypeptide sequence of a telomerase and thefusion protein has telomerase activity in a cell treated with tamoxifenor a tamoxifen derivative. Non-limiting exemplary sequences of histoneacetyltransferase, histone deactyltransferase, O-6-methylguanine-DNAmethyltransferase, and telomerase that can be present in an ER fusionprotein are listed in Table 2 below. Methods for detecting genetranscription, histone deacetylase activity, histone acetyltransferaseactivity, O-6-methylguanine-DNA methyltransferase activity, andtelomerase activity are known in the art.

TABLE 2 Exemplary Fusion Partner Polypeptides Histone NCBI AccessionNos. AAD42348.1 and AF140360.1 Acetyltransferase Histone NCBI AccessionNos. AAQ18232.1 and AY302468.1 Deacetylase O-6- NCBI Accession Nos.AAA59594.1 and M60761.1 Methylguanine- DNA Methyltransferase TelomeraseNCBI Accession Nos. NP_937983.2, NM_198253.2, NP_001180305.1, andNM_001193376.1

In some embodiments, the fusion partner polypeptide contains apolypeptide sequence of a viral protein (e.g., an adenoviral,lentiviral, or a retroviral protein). In some embodiments, the fusionpartner polypeptide contains a polypeptide sequence from a pathogenicbacterium or a parasite. In some embodiments, the fusion partnerpolypeptide contains a polypeptide sequence of an oncogene (e.g., ahuman, mouse, rat, or monkey oncogene). Additional sequences of fusionpartner polypeptides are described in the examples and are known in theart.

In some embodiments, the ER fusion proteins can contain a polypeptidesequence of an additional reporter protein (e.g., a fluorescent proteinsuch as green fluorescent protein or yellow fluorescent protein, or anenzyme capable of producing a colored product (e.g., β-galactosidase) oran additional epitope (e.g., a His-tag).

Promoters

In some embodiments, a nucleic acid encoding the ER fusion protein(e.g., any of the ER fusion proteins described herein) is stablyintegrated into a chromosome in the nucleus of a eukaryotic (e.g.,mammalian, plant, yeast, nematode, parasite, or reptile) cell. In someembodiments, a nucleic acid encoding the ER fusion protein is introducedinto a chromosome of the eukaryotic (e.g., mammalian, plant, yeast,nematode, parasite, or reptile) cell using a recombinant virus (e.g., arecombinant adenovirus, lentivirus, or retrovirus). In some embodiments,a nucleic acid encoding the ER fusion protein is introduced into anembryo or blastocyst of non-human mammal to produce a transgenic mammal.

In some embodiments, a nucleic acid encoding the ER fusion protein(e.g., any of the ER fusion proteins described herein) is located in anexpression plasmid or an artificial mammalian chromosome (e.g., a humanartificial chromosome), and the expression plasmid or artificialmammalian chromosome is introduced into a mammalian cell (e.g., amammalian embryo or blastocyst). In some embodiments, the eukaryoticcell is a yeast cell and a yeast artificial chromosome is introducedinto the yeast cell. In some embodiments, the expression plasmid isintroduced into the eukaryotic cell by electroporation or lipofection.In some embodiments, the artificial chromosome is introduced into theeukaryotic cell by microinjection or cell fusion (e.g., fusion of atarget cell (e.g., an embryo) with a cell (e.g., a fetal fibroblast)containing a mammalian artificial chromosome). A variety of differenteukaryotic (e.g., mammalian) expression plasmids and mammalianartificial chromosomes are known in the art. A variety of differentmolecular biology methods for introducing an expression plasmid or amammalian artificial chromosome into a eukaryotic cell are also known inthe art.

In some embodiments, one or more promoter or enhancer sequences can beoperatively linked (e.g., upstream) to the sequence encoding the ERfusion protein. In some embodiments, the one or more promoter sequencescan be a tissue-specific promoter, an inducible e promoter, or aubiquitous (e.g., strong ubiquitous) promoter. Non-limiting examples oftissue-specific promoters include B29 promoter (B-cell expression), CD14promoter (monocyte expression), CD43 promoter (leukocyte and plateletexpression), CD45 promoter (haematopoietic cell expression), CD68promoter (macrophage expression), desmin promoter (muscle cellexpression), elastase-1 promoter (pancreatic acinar cell expression),endoglin promoter (endothelial cell expression), fibronectin promoter(differentiating cell expression), Flt-1 promoter (endothelial cellexpression), GFAP promoter (astrocyte expression), GPIIB promoter(megakaryocyte expression), ICAM-2 promoter (endothelial cellexpression), INF-β promoter (hematopoietic cell expression), Mb promoter(muscle cell expression), NphsI promoter (podocyte expression), OG-2promoter (osteoblast expression), SP-B promoter (lung cell expression),SYN1 promoter (neuron expression), WASP promoter (hematopoietic cellexpression), SV40/bAlb promoter (liver cell expression), and NSE/Ru5′promoter (mature neuron expression) (these promoter sequences areavailable in commercially available vectors, e.g., expression vectorsavailable from InvivoGen). In some embodiments, an aromatase promoter isused to drive expression of the ER fusion protein in breast tissue (see,e.g., Khan et al., Reprod. Biol. Endocrinol. 9:91, 2011). In someembodiments, a carbonic anhydrase I promoter/enhancer is used to expressthe ER fusion protein in colon tissue (see, e.g., Xue et al., Mol.Cancer Res. 8:1095, 2010). In some embodiments, a keratin 14 promoter isused to express the ER fusion protein in skin tissue (see, e.g.,Vandermeulen et al., Vaccine 27:4272-4277, 2009). Additional examples oftissue-specific promoter sequences are known in the art.

In some embodiments, the nucleic acid encoding the ER fusion protein isoperatively linked (e.g., upstream) to an inducible promoter sequenceand the cell is further contacted with a chemical inducing agent.Non-limiting examples of inducible promoter sequences includetetracycline-regulated promoters (see, e.g., the T-Rex™ system from LifeTechnologies), doxycycline-inducible promoters (see, e.g., Qin et al.,PLoS ONE 5:e10611, 2010), RU486-inducible promoters (see, e.g., U.S.Patent Application Publication No. 2009/0293139), andpolyinosinic:polycytidylic acid (Poly(I:C))-inducible promoters (Tomitaet al., Mol. Endocrinol. 14:1674-1681, 2000). Additional induciblepromoter sequences and chemical inducing agents are known in the art.

In some embodiments, the nucleic acid encoding the ER fusion protein isoperatively linked (e.g., upstream) to a ubiquitous (e.g., a strongubiquitous) promoter sequence. Non-limiting examples of ubiquitouspromoters include β-actin promoter, CMV promoter, SV40 promoter, UBCpromoter, EF1A promoter, PGK promoter, and CAGG promoter (see, e.g., Qinet al., PLoS One 5:e10611, 2010). One or more of the promoter sequencesdescribed herein can be operatively linked to a nucleic acid encoding anER fusion protein using molecular biology methods known in the art.

Methods of Inducing Nuclear Translocation

Provided herein are methods of optically inducing nuclear translocationof a fusion protein containing a mammalian estrogen receptor ligandbinding domain (e.g., a human estrogen receptor ligand binding domain)in a eukaryotic (e.g., mammalian, plant, yeast, nematode, parasite, orreptile) cell. These methods include providing a eukaryotic ((e.g.,mammalian, plant, yeast, nematode, parasite, or reptile) cell thatcontains a fusion protein containing a mammalian (e.g., human) estrogenreceptor ligand binding domain, contacting the cell with any of thecompositions described herein; and irradiating the cell with awavelength of light between of between 200 nm and 900 nm (e.g., between350 nm to 410 nm, between 200 nm and 700 nm, and between 700 nm and 900nm) for a period of time sufficient to stimulate release of tamoxifen ora tamoxifen derivative from the composition, where the releasedtamoxifen or tamoxifen derivative induces the nuclear translocation ofthe fusion protein. In some embodiments, the fusion protein is any ofthe fusion proteins (ER fusion proteins) described herein.

The nuclear translocation of a fusion protein can be detected usingimmunofluorescence using antibodies that bind specifically to either themammalian estrogen receptor ligand binding domain or the fusion partnerpolypeptide. The nuclear translocation of the fusion protein can also beassessed by detecting the activity of the fusion protein in the nucleusof the cell (e.g., transcription factor activity, transcriptionrepressor activity, telomerase activity, oncogene activity, histonedeacetylase activity, histone acetyltransferase activity, orO-6-methylguanine DNA methyltransferase activity in the nucleus of thecell). In some embodiments, the fusion protein can further contain adetectable reporter protein (e.g., a green fluorescent protein or yellowfluorescent protein) that can be used to detect the presence of thefusion protein in the nucleus of the cell.

In some embodiments, the eukaryotic (e.g., mammalian, plant, yeast,nematode, parasite, or reptile) cell contains a nucleic acid encodingthe fusion protein. In some embodiments, the nucleic acid encoding thefusion protein is stably integrated in a chromosome within the nucleusof the eukaryoatic (e.g., mammalian, plant, yeast, nematode, parasite,or reptile) cell. In some embodiments, the nucleic acid encoding thefusion protein is located in an expression plasmid or a mammalianartificial chromosome (e.g., a human artificial chromosome). In someembodiments, the nucleic acid encoding the fusion protein is operablylinked to one or more promoter or enhancer sequences. In someembodiments, the nucleic acid is operatively linked to a induciblepromoter (e.g., any of the inducible promoters described herein or knownin the art) and the cell is further contacted with a chemical inducingagent (e.g., any of the chemical inducing agents described herein orknown in the art) (e.g., prior to contacting with the caged tamoxifen orcaged tamoxifen derivative molecule, prior to contacting with the cagedtamoxifen or caged tamoxifen derivative molecule and prior toirradiation, or after contacting with the caged tamoxifen or cagedtamoxifen derivative molecule and after irradiation). In someembodiments, the nucleic acid encoding the fusion protein is operativelylinked to a tissue-specific promoter (e.g., any of the tissue-specificpromoters described herein or known in the art). In some embodiments,the nucleic acid encoding the fusion protein is operatively linked to aubiquitous promoter (e.g., any of the ubiquitous promoters describedherein or known in the art).

In some embodiments, the eukaryotic (e.g., mammalian, plant, yeast,nematode, parasite, or reptile) cell is present in vitro or ex vivo. Insome embodiments, the eukaryotic (e.g., mammalian) cell is an epithelialcell, a lung cell, a kidney cell, an endothelial cell, a muscle cell, anadipose cell, a bone cell, a cartilage cell, or a neuron. In someembodiments, the eukaryotic (e.g., mammalian) cell is anundifferentiated cell or a stem cell (e.g., an embryonic stem cell) andthe fusion protein contains a sequence of a transcription factor or atranscription repressor that induces cellular differentiation. In someembodiments, the eukaryotic (e.g., mammalian) cell is implanted into amammal following the contacting with the caged tamoxifen or cagedtamoxifen derivative molecule and irradiation.

In some embodiments, the cell is present in a mammal (e.g., a mouse orrat). In some embodiments, the cell is present in the breast tissue,skin, kidney, lung, colon, liver, pancreas, stomach, intestine, colon,muscle, mammary gland, ovary, testes, prostate, brain, spinal cord,peripheral nerve, and heart of the mammal. In some embodiments, themammal is a child. In some embodiments, the mammal is an adult. In someembodiments, the mammal is an embryo or blastocyst.

In some embodiments, the composition is locally administered to a tissuecontaining the cell. In some embodiments, the composition issystemically administered (e.g., intravenous, intraarterial,intramuscular, intraperitoneal, subcutaneous, oral, transdermal, ocular,nasal, or intrathecal administration). In some embodiments, more thanone dose of the composition is administered to the mammal before thecell is irradiated. In some embodiments, the cell can be contacted witha dose of the composition and then irradiated several times (e.g., oneor more (e.g., at least two, three, four, or five) rounds of contactingand irradiating can be performed).

In some embodiments, a mammal is administered a dose of between 1 mg to500 mg of the composition (e.g., between 1 mg to 400 mg, between 1 mg to300 mg, between 1 mg and 250 mg, between 1 mg and 200 mg, between 1 mgand 150 mg, between 1 mg and 100 mg, between 1 mg and 50 mg, between 5mg and 50 mg, and between 5 mg and 40 mg).

In some embodiments, the fusion protein contains a sequence of arecombinase (e.g., any of the recombinases described herein or known inthe art), and the fusion protein has recombinase activity. In someembodiments, the fusion protein contains a sequence of a transcriptionfactor, and the transcription factor is capable of promoting genetranscription in the nucleus of the cell. In some embodiments, thefusion protein promotes gene transcription of a gene that is present ina recombinant gene construct that is stably integrated in a chromosomepresent in the nucleus of the cell. In some embodiments, the fusionprotein promotes transcription of an endogenous gene. Methods ofdetermining the ability of a fusion protein to promote genetranscription are known in the art (e.g., reverse-transcriptasereal-time polymerase chain reaction (PCR)).

In some embodiments, the fusion protein contains a sequence of atranscription repressor, and the fusion protein is capable of repressinggene transcription in the nucleus of the cell. In some embodiments, thefusion protein represses gene transcription of a gene that is present ina recombinant gene construct that is stably integrated in a chromosomepresent in the nucleus of the cell. In some embodiments, the fusionprotein represses gene transcription of an endogenous gene. Methods ofdetermining the ability of a fusion protein to repress genetranscription are known in the art (e.g., reverse transcriptasereal-time PCR).

In some embodiments, the fusion protein contains a sequence of a histonedeacetylase, a histone acetyltransferase, or an O-6-methylguanine DNAmethyltransferase, and the fusion protein has histone deacetylase,histone acetyltransferase, or O-6-methylguanine DNA methyltransferaseactivity, respectively. In some embodiments, the fusion protein containsa sequence of a telomerase, and the fusion protein has telomeraseactivity. Methods for determining the histone deacetylase, histoneacetyltransferase, O-6-methylguanine DNA methyltransferase, ortelomerase activity are known in the art.

In some embodiments, the fusion protein contains a sequence of anoncogene.

In some embodiments, the cell is irradiated for a period of at least 1minute (e.g., at least 5, 10, 15, 20, 30, 40, 50, or 60 minutes). Insome embodiments, the cell is illuminated for a maximum of 15, 20, 25,30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the cell isilluminated using any of the light sources described herein (e.g., ahand-held light source). In some embodiments, the contacting and theirradiating occur within 24 hours of each other (e.g., within 20, 16,12, 10, 8, 6, 4, 3, 2, or 1 hour of each other).

In some embodiments of all of these methods, the composition containsDMNPE-conjugated 4-hydroxycyclofen and the cell is irradiated with 350nm to 410 nm light.

In some embodiments of all of these methods, the cell is irradiated withlight of between 700 nm and 800 nm, between 700 nm and 900 nm, between650 nm and 850 nm, between 500 nm and 800 nm, or between 600 nm and 900nm.

Methods of Inducing Recombination

Also provided are methods of optically inducing recombination in aeukaryotic (e.g., mammalian, plant, yeast, nematode, parasite, orreptile) cell. These methods include: providing a eukatyotic (e.g.,mammalian, plant, yeast, nematode, parasite, or reptile) cell thatcontains (i) a nucleic acid encoding a fusion protein containing asequence of a recombinase and a sequence of a human estrogen receptorligand binding domain, where the fusion protein has recombinaseactivity, and (ii) a recombinase recognition sequence that isspecifically recognized by the fusion protein, where both the nucleicacid encoding the fusion protein and the recombinase recognitionsequence are integrated into a chromosome within the nucleus of thecell; contacting the cell with any of the compositions described herein;and irradiating the cell with a wavelength of light between 200 nm to900 nm (e.g., between 350 nm to 410 nm, between 200 nm and 700 nm, andbetween 700 nm and 900 nm) for a period of time sufficient to stimulaterelease of tamoxifen or a tamoxifen derivative from the composition,where the tamoxifen or tamoxifen derivative stimulates the nucleartranslocation of the fusion protein and the fusion protein stimulatesrecombination at the recombinase recognition sequence. In someembodiments, the cell is irradiated with light between 700 nm and 800nm, between 700 nm and 900 nm, between 650 nm and 850 nm, between 500 nmand 800 nm, or between 600 nm and 900 nm.

In some embodiments, the nucleic acid encodes a fusion proteincontaining a sequence of any of the recombinases described herein (e.g.,a Cre recombinase). As is known in the art, each recombinase recognizesa unique recombinase recognition sequence. For example, Cre recombinaserecognizes a 34-base pair loxP recombinase recognition sequence.

In some embodiments, the nucleic acid encoding the fusion protein isoperably linked to one or more promoter or enhancer sequences. In someembodiments, the nucleic acid is operatively linked to a induciblepromoter (e.g., any of the inducible promoters described herein or knownin the art) and the cell is further contacted with a chemical inducingagent (e.g., any of the chemical inducing agents described herein orknown in the art) (e.g., prior to contacting with the caged tamoxifen orcaged tamoxifen derivative, prior to contacting with the caged tamoxifenor caged tamoxifen derivative and prior to irradiation, or aftercontacting with the caged tamoxifen or caged tamoxifen derivative andafter irradiation). In some embodiments, the nucleic acid encoding thefusion protein is operatively linked to a tissue-specific promoter(e.g., any of the tissue-specific promoters described herein or known inthe art). In some embodiments, the nucleic acid encoding the fusionprotein is operatively linked to a ubiquitous promoter (e.g., any of theubiquitous promoters described herein or known in the art).

In some embodiments, the nucleic acid encoding the fusion protein andthe recombinase recognition sequence are located on differentchromosomes in the nucleus of the cell. In some embodiments, therecombination results in a decrease in the expression of a transgenelocated between two recombinase recognition sequences in the chromosome.In some embodiments, the recombination results in the replacement of asequence between two recombinase recognition sequences with a newtransgenic sequence. In some embodiments, the recombination results inthe increase in the proximity of a promoter or enhancer sequence to atransgene, wherein the recombination results in increased expression ofthe transgene. In some embodiments, the recombination results in theincrease in the proximity of a repressor sequence or heterochromatin toa transgene, wherein the recombination results in decreased expressionof the transgene.

In some embodiments, the removal of a sequence present between tworecombinase recognition sequences results in an increase in theexpression of a proximal transgene or proximal homologous gene. In someembodiments, removal of a sequence between two recombinase recognitionsequences results in a decrease in the expression of a proximaltransgene or a proximal homologous gene.

In some embodiments, the eukaryotic (e.g., mammalian, plant, yeast,nematode, parasite, or reptile) cell is present in vitro or ex vivo. Insome embodiments, the eukaryotic (e.g., mammalian) cell is an epithelialcell, a lung cell, a kidney cell, an endothelial cell, a muscle cell, anadipose cell, a bone cell, a cartilage cell, or a neuron. In someembodiments, the eukaryotic (e.g., mammalian) cell is anundifferentiated cell or a stem cell (e.g., an embryonic stem cell). Insome embodiments, the cell is implanted into a mammal following thecontacting with the caged tamoxifen or caged tamoxifen derivativemolecule and irradiation.

In some embodiments, the cell is present in a mammal (e.g., a mouse orrat). In some embodiments, the cell is present in the breast tissue,skin, kidney, lung, colon, liver, pancreas, stomach, intestine, colon,muscle, mammary gland, ovary, testes, prostate, brain, spinal cord,peripheral nerve, and heart. In some embodiments, the mammal is a child.In some embodiments, the mammal is an adult. In some embodiments, themammal is a female. In some embodiments, the mammal is a male. In someembodiments, the mammal is an embryo or blastocyst.

In some embodiments, the composition is locally administered to a tissuecontaining the cell. In some embodiments, the composition issystemically administered (e.g., intravenous, intraarterial,intramuscular, intraperitoneal, subcutaneous, oral, transdermal, ocular,nasal, or intrathecal administration). In some embodiments, more thanone dose of the composition is administered to the mammal before thecell is irradiated. In some embodiments, the composition is systemicallyadministered and a specific target tissue is irradiated. In someembodiments, the composition is locally administered to a target tissueand the target tissue is irradiated.

In some embodiments, a mammal is administered a dose of between 1 mg to500 mg of the composition (e.g., between 1 mg to 400 mg, between 1 mg to300 mg, between 1 mg and 250 mg, between 1 mg and 200 mg, between 1 mgand 150 mg, between 1 mg and 100 mg, between 1 mg and 50 mg, between 5mg and 50 mg, and between 5 mg and 40 mg).

In some embodiments, the cell is irradiated for a period of at least 1minute (e.g., at least 5, 10, 15, 20, 30, 40, 50, or 60 minutes). Insome embodiments, the cell is irradiated for a maximum of 15, 20, 25,30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the cell isirradiated using any of the light sources described herein (e.g., ahand-held light source). In some embodiments, the cell is irradiatedusing an endoscopic or orthoscopic procedure. In some embodiments, thecell is irradiated using a two-photon light source. In some embodiments,the contacting and the irradiating occur within 24 hours of each other(e.g., within 20, 16, 12, 10, 8, 6, 4, 3, 2, or 1 hour of each other).

Methods of Treatment of Breast Cancer

Also provided are methods of treating a breast cancer in a mammal (e.g.,human) that include administering a therapeutically effective amount ofa composition containing a caged tamoxifen or a caged tamoxifenderivative (e.g., any of the caged tamoxifen or caged tamoxifenderivative molecules described herein), and irradiating a mammary tissuewith light of between 200 nm to 900 nm (e.g., between 350 nm to 410 nm,between 200 nm and 700 nm, and between 700 nm and 900 nm), wherein theirradiating results in the release of tamoxifen or a tamoxifenderivative in the mammary tissue of the subject.

In some embodiments, the composition contains

In some embodiments, the composition is formulated for sustained-release(e.g., formulated in a biodegradable polymer or a nanoparticle). In someembodiments, the composition is administered locally to the mammarytissue of the subject (e.g., local intraglandular, periglandular,subcutaneous, interductal, transdermal, or intramuscularadministration). In some embodiments, the composition is administeredsystemically (e.g., oral, intravenous, intraarterial, intraperitoneal,intramuscular, or subcutaneous administration). In some embodiments, thecomposition is formulated for oral, intraglandular, periglandular,subcutaneous, interductal, intramuscular, intraperitoneal,intramuscular, intraarterial, transdermal, or intravenousadministration).

In some embodiments, the subject is a female. In some embodiments, thesubject is a male. In some embodiments, the subject is a child. In someembodiments, the subject is an adult (e.g., at least 18, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 years old). In someembodiments, the subject has been diagnosed as having breast cancer. Insome embodiments, the subject has been resistant to other breast cancertherapeutics.

A subject can be identified as having breast cancer by the detection orobservation of one of more the following symptoms in a subject: breastlump or thickening, bloody discharge from a nipple, change in the sizeor shape of breast, changes to the skin over the breast, invertednipple, peeling, flaking, or scaling of a nipple or breast skin, rednessor pitting of the skin over the breast, mutations in the Her2/neureceptor, and mutations in BRCA1 or BRCA2. A subject can be diagnosed ashaving breast cancer by a medical professional (e.g., a physician, anurse, a nurse's assistant, a physician's assistant, or a laboratorytechnician). The efficacy of a treatment of a breast cancer can bedetected by a decrease in the size of a breast cancer tumor, a decreasein the spread of the breast cancer in the mammary tissue of the subject,a decrease in the rate of metastasis of breast cancer in the subject(e.g., as compared to a subject not receiving a treatment or receiving adifferent treatment for breast cancer), a decrease in the rate of growthof a breast cancer tumor in a subject, and a decrease in one or morephysical symptoms of breast cancer (e.g., a decrease in one or more ofthe physical symptoms listed above).

In some embodiments, the subject is administered a dose of between 1 mgto 500 mg of any of the compositions described herein (e.g., between 1mg to 400 mg, between 1 mg to 300 mg, between 1 mg and 250 mg, between 1mg and 200 mg, between 1 mg and 150 mg, between 1 mg and 100 mg, between1 mg and 50 mg, between 5 mg and 50 mg, and between 5 mg and 40 mg). Theamount of the composition administered will depend on whether thecomposition is administered locally or systemically. A skilled artisancan further determine the appropriate dosage depending, for example, onthe following factors: whether the administration is local or systemic,the age of the subject, the severity or stage of the disease, the othertherapies administered to the subject, the subject's mass, the subject'ssex, and the subject's responsiveness to other breast cancertherapeutics.

In some embodiments, the subject is administered more than one dose ofthe composition. In some embodiments, the subject is administered a doseof the composition at least once a month (e.g., at least twice a month,at least three times a month, at least four times a month, at least oncea week, at least twice a week, three times a week, once a day, or twicea day). In some embodiments, the mammary tissue of the subject isirradiated within 24 hours (e.g., within 20, 16, 12, 10, 8, 6, 4, 2, or1 hour) of the administration of the composition. In some embodiments,the mammary tissue of the subject is irradiated within 30 minutes (e.g.,within 20 minutes, 15 minutes, 10 minutes, or 5 minutes) of theadministration of the composition.

In some embodiments, the mammary tissue is irradiated for a period of atleast 1 minute (e.g., at least 5, 10, 15, 20, 30, 40, 50, or 60minutes). In some embodiments, the mammary tissue is irradiated for amaximum of 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In someembodiments, the mammary tissue is irradiated using any of the lightsources described herein (e.g., a hand-held light source). In someembodiments, the mammary tissue is irradiated using two-photon lightsource.

In some embodiments, serial rounds of contacting and irradiating can beperformed in the subject (e.g., at least two rounds ofcontacting/irradiating). In some embodiments, the rounds ofcontacting/irradiating can be performed periodically (e.g., at leastonce a week, at least a month, at least once every two months, at leastonce every three months) in the subject over an extended period of time(e.g., a period of at least 2 weeks, at least one month, at least twomonths, at least six months, at least one year, and at least two years).

In some embodiments, the administering and irradiating are performed bya health care professional (e.g., a nurse, a nurse's assistant, aphysician, a physician's assistant, or a laboratory technician). In someembodiments, the administering and irradiating are performed by thesubject.

In some embodiments, the subject is administered one or more additionalbreast cancer therapeutics. In some embodiments, the one or moreadditional breast cancer therapeutics are selected from the groupconsisting of: cyclophosphamide, methotrexate, 5-fluorouracil,doxorubicin, paclitaxel, doxorubicin, epirubicin, trastuzumab,lapatinib, bevacizumab, abraxane, pamidronate, anastrozole, exemastane,fulvestrant, letrozole, gemcitabine, pegfilgrastim, filgrastin,doxetaxel, capecitabine, goserelin, zoledronic acid, and ixabepilone. Insome embodiments, the compositions contain a caged tamoxifen or cagedtamoxifen derivative molecule (e.g., any of the caged tamoxifen or cagedtamoxifen derivative molecules described herein) and one or moreadditional breast cancer therapeutics (e.g., any of the additionalbreast cancer therapeutics described herein).

The invention is further described in the following example, which doesnot limit the scope of the invention described in the claims.

Example Example 1. Synthesis and Use of Caged 4-OHC to Achieve PreciseGenetic Engineering Control in Mice

In order to achieve cell-specific gene activation, a caged tamoxifenderivative molecule was synthesized and tested in a series of in vitroand in vivo photoactivation experiments to determine whether it couldreliably induce efficient light-dependent Cre-mediated recombination inmice. The methods and materials used in these experiments are describedbelow.

Materials and Methods

Reagents and Instrument

Unless otherwise stated, all the reagents for the synthesis of caged4-OHC were obtained from Sigma-Aldrich (St. Louis, Mo., USA) and used asreceived. ¹H NMR spectra were recorded on a Varian 400 MHz spectrometer.High performance liquid chromatography-mass spectrometry (HPLC-MS)analysis was performed on a Waters (Milford, Mass.) LC-MS system. In theLC-MS system, electrospray ionization (ESI) was used to obtain massspectrometry. A Waters XTerra C18 5-μm column was used for HPLC-MSanalysis (eluents: 0.1% trifluoroacetic acid (v/v) in water andacetonitrile; gradient: 0-9.5 min, 5-100% B; 9.5-10.0 min 100% B). Thechromatograms were processed using MassLynx software (from Waters).UV-Vis spectra were recorded in a TECAN microplate reader. A 6 Whand-held UV lamp (UVP, LLC) was used for uncaging the caged 4-OHC bothin vitro and in vivo.

Mice

Homozygous Rosa26CreER^(T2) (Ventura et al., Nature 445:661-665, 2007),mT/mG (Muzumdar et al., Genesis 45:593-605, 2007), and R26R (Soriano etal., Nat. Genetics 21:70-71, 1999) mice in C57BL/6 background wereobtained from The Jackson Laboratory (Stock Numbers: 8463, 7676 and3474, respectively). Heterozygous Rosa26CreER^(T2);mT/mG andRosa26CreER^(T2);R26R mice were generated by crossing the homozygousmice.

Synthesis of Caged 4-OHC

The DMNPE photoactivatable caging group and 4-OHC were synthesized asdescribed (Sinha et al., Chembiochem 11:653-663, 2010; Dyer et al., J.Org. Chem. 64:7988-7995, 1999). DMNPE was conjugated to 4-OHC underMitsonobu coupling conditions. Briefly, in a 25-mL round bottom flask,DMNPE (0.014 g, 0.063 mmol), 4-OHC (0.020 g, 0.057 mmol), and triphenylphosphine (PPh3, 0.016 g, 0.063 mmol) were mixed together in 0.5 mL oftetrahydrofuran (THF) under an argon atmosphere. After stirring thesolution at room temperature (RT) for ˜5 min, diisopropylazodicarboxylate (DIAD, 0.012 ml, 0.063 mmol) was added drop-wise to thereaction mixture. The reaction mixture was allowed to stir at RT for˜2.5 h. The crude product was directly charged to a Sift column forpurification (eluent: 100% dichloromethane to 10% methanol indichloromethane v/v). Caged 4-OHC was isolated as a yellow solid.Yield=31%. ¹H NMR (400 MHz, CDCl₃): 7.65 (s, 1H), 7.20 (s, 1H), 6.93 (m,4H), 6.79 (d, ²J=8.8 Hz, 2H), 6.67 (d, ²J=8.8 Hz, 2H), 6.10 (q, ⁴J=6.13Hz, 1H), 4.03 (t, ³J=5.8 Hz, 2H), 3.94 (s, 3H), 3.89 (s, 3H), 2.72 (t,³J=5.8 Hz, 2H), 2.35 (s, 6H), 2.18 (m, 4H), 1.67 (d, ²J=6 Hz, 3H), 1.56(m, 6H). MS (electrospray ionization mass spectrometry: ESI-MS)calculated: 560.29, found: 561.40 [M+H]⁺.

UV-Vis and HPLC-MS Characterization of the Photocleavage of Caged 4-OHC

A 0.25 mM solution of caged 4-OHC in 1:1 (v/v) water:acetonitrile wasused for the UV-Vis spectroscopic characterization of the photocleavagereaction. The solution of the caged 4-OHC was placed in a 96-well blackclear bottom microplate. The solution was then irradiated at ˜365 nmusing a hand-held UV lamp. After light exposure, UV-Vis spectra of thesolution was recorded in a TECAN microplate reader. A time course of thephotocleavage reaction was monitored by exposing the caged 4-OHCsolution to light for different durations, and subsequently recordingthe UV-Vis spectra of the solution.

A 2.0 mM solution of caged 4-OHC in 1:1 (v/v) water:acetonitrile wasused for the HPLC-MS study. The solution of the caged 4-OHC was placedin a glass vial and irradiated at ˜365 nm using a hand-held UV lamp.Aliquots were taken at different time intervals and injected to theHPLC-MS machine for analysis. Prism 5 (GraphPad, La Jolla, Calif.) forMac was used to plot the data.

MEF Isolation and Photoactivation Procedure

Mouse embryonic fibroblasts (MEFs) were isolated as described (Sharplesset al., Mol. Cell 8:1187-1196, 2001) and grown in DMEM with 10% FBS. Toshed UV light on the cells, the cells were first grown in 60-mm dishovernight to become nearly confluent. The next day, the medium wasreplaced with medium containing caged 4-OHC at 5 μM and incubated for 30minutes. The cells were washed twice quickly with warm PBS and coveredwith fresh medium. The dish was placed on the UV emission surface of ahandheld 6 W long-wavelength UV lamp (UVP, LLC), and UV light was kepton for a designated period of time. The cells were put back into theincubator to culture for 48-72 h before live imaging or flow cytometricanalysis.

Mammary 3D Culture and Photoactivation Procedure.

Mouse mammary epithelial cells (MEFs) were isolated from 6-8 week oldfemale Rosa26CreER^(T2);mT/mG mice as previously described (Tiede etal., PLoS One 4:e8035, 2009). Briefly, mammary glands were excised,minced using scalpels, and digested for 1 hour in 300 U/mL type 1Acollagenase (Sigma) and 100 U/mL hyaluronidase (Sigma). The cells werethen treated with 0.25% trypsin/EDTA, dispase (Invitrogen)/DNase(Sigma), and ACK lysing buffer (Invitrogen) in succession. Between eachtreatment, cells were rinsed in MEGM (1:1 DMEM:F12 Ham supplemented with5 mg/mL insulin, 500 ng/mL hydrocortisone, 10 ng/mL EGF, 20 ng/mLcholera toxin, 5% bovine calf serum, and 1× penicillin/streptomycin).Afterwards, cells were filtered twice through 40-mm nylon cell strainersand seeded in 35-mm dishes that contained a layer of Growth FactorReduced Matrigel (BD Biosciences) measuring approximately 1-2 mmthickness. Acini usually formed in 4-8 days. During photoactivation, theacini were incubated with MEGM containing 5 μM caged 4-OHC for 1 hour.The cells were washed twice quickly with warm PBS and covered with freshmedium. The dish was placed on the UV emission surface of a hand-held 6W long-wavelength UV lamp (UVP, LLC, exposure time: 1 minute) or abovethe 60× objective of an inverted epifluorescent microscope equipped witha standard DAPI filter set. The cells were returned to the incubator toculture for >48 h before live-cell imaging using an upright Zeiss 710laser scanning confocal microscope equipped with 20× and 40× waterimmersion objectives.

Photoactivation Procedure in Mice

Female mice of 6-8 weeks old were clean shaven on the dorsal and ventralsides. The caged 4-OHC was dissolved in 20% Solutol for in vivodelivery. The vehicle or 1 mg caged 4-OHC in vehicle was injectedintraperitoneally. One hour later, the mice were anesthetized withisoflurane and exposed to UV light from the hand-held 6 Wlong-wavelength UV lamp (UVP, LLC) for 15 minutes. Alternatively, formammary tissue illumination, the right inguinal (#4) mammary fat pad wasexposed by creating a small skin flap and illumination with UV light for15 minutes. For enhanced photoconversion, this procedure was repeatedfour times. On the seventh day, the mice were imaged with Olympus OV-110epifluorescence imager (Thurber et al., PLoS One 4:e8053, 2009) todetect fluorescent signals emitted from the skin on the dorsal andventral sides. Alternatively, the left and right mammary glands weredissected and immediately imaged using the intravital laser scanningmicroscope IV-110 (Kelly et al., PLoS Med. 5:e85, 2008).

Results

A recently developed double-fluorescent Cre reporter mouse, the mT/mGstrain (Muzumdar, Genesis 45:593, 2007) was used to design a biologicalsystem that can faithfully report on the induced activity of CreER. Thismouse model expresses tdTomato prior to and EGFP following Cre-mediatedrecombination ubiquitously in tissues. The homozygous mT/mG mouse wascrossed to the homozygous Rosa26CreER^(T2) strain (Ventura et al.,Nature 445:661, 2007), and the progeny, Rosa26CreER^(T2);mT/mG, wereheterozygous for both alleles. The photoinduced activity of cagedtamoxifen and caged tamoxifen derivative molecules can be assessed byilluminating the cells and tissues from these mice and looking forEGFP-expressed cells (FIG. 1).

In these experiments, a 4-hydroxytamoxifen (4-OHT) analogue,4-hydroxycyclofen (4-OHC), was used as a small molecule agonist of theER component of the fusion protein. Although 4-OHT and 4-OHC havesimilar binding affinity to the ER, 4-OHC is preferred over 4-OHT inview of its synthetic accessibility and better photostability (Zinha etal., Zebrafish 7:199, 2010). As shown in FIG. 2, 4-OHC was caged byattaching a photolabile 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE)moiety to the free hydroxy group of 4-OHC using the Mitsonobu reaction(Zinha et al., Zebrafish 7:199, 2010). Under ambient light, the DMNPEcaging group is stable in physiological conditions. However, exposure tolong wavelength UV irradiation (˜350-410 nm with a peak at 365 nm) leadsto photolytic cleavage, releasing 4-OHC (FIG. 3). The photocleavagereaction of the caged 4-OHC was monitored by UV-Vis spectroscopy. Achange in the UV-Vis absorption spectrum was observed when a solution ofcaged 4-OHC was exposed to a hand-held 365 nm UV lamp. This change wastypical of the breakage of the DMNPE caging group (FIG. 4). Thephotochemical reaction was complete within 10 min of exposure (FIG. 5).High performance liquid chromatography-mass spectrometry (HPLC-MS)verified the chemical identity of the photoreleased products and alsoconfirmed quantitative, unidirectional conversion to 4-OHC (FIG. 6).Overall, these characterizations indicate that the caged 4-OHC undergoesefficient photocleavage at 365 nm UV light and releases 4-OHC.

Mouse embryonic fibroblasts (MEFs) isolated from theRosa26CreER^(T2);mT/mG mice in cell culture were used to test the caged4-OHC activity. The Rosa26CreER^(T2);mT/mG MEFs were treated with either4-OHC or caged 4-OHC and illuminated at 365 nm. As expected,photocleavage of the caged 4-OHC induced EGFP expression in a stringentfashion (FIGS. 7 and 8). To further test the caged 4-OHC, anotherreporter mouse was generated (Rosa26CreER^(T2);R26R mice). TheRosa26CreER^(T2);R26R mice were generated by crossing homozygousRosa26CreER^(T2) mice with homozygous Rosa26-loxP-STOP-loxP-lacZ mice(experimental system shown in FIG. 9). Similar results were observed inthe Rosa26CreER^(T2);R26R MEFs (FIG. 10). No significant phototoxicityor cell viability changes due to the UV irradiation was observed inthese experiments (energy density 1.6 mW/cm², photon energy 3.4 eV,number of photons per second per cm² 2×10¹⁵, up to 3 min exposure time),consistent with other reports (Young et al., Org. Biomol. Chem. 5:999,2007).

While a convenient system, 2D-cell culture cannot manifest all thebiological responses of cells to external perturbations in a3D-environment. Additional tests were performed in mammary acinusculture to determine whether caged 4-OHC would enable light-dependentCre recombination in this well-established organoid model. Mammaryepithelial cells from Rosa26CreER^(T2);mT/mG mice were isolated andoverlaid on a basement membrane to allow polarized acinar structuredevelopment. The formed acini were subjected to caged 4-OHC treatmentwith or without subsequent brief UV illumination by the UV lamp (energydensity 1.6 mW/cm², photon energy 3.4 eV, number of photons per secondper cm²2×10¹⁵, 1 min exposure time) (FIG. 11) or the 60× objective of aninverted fluorescent microscope equipped with a standard DAPI filter set(FIG. 12). Similar to the 2D-culture results, the caged 4-OHC allowedvery tight control of EGFP expression in response to photoactivation inthe mammary acini experimental model.

Additional experiments were performed to determine whether caged 4-OHCis able to induce gene activation in vivo in mice upon photoactivation.These experiments focused on two organs: skin and mammary glands. Acustom-built broad illumination method was used to perform this in vivotesting. In a first set of experiments, EGFP/tdTomato expression at thewhole mouse level was assessed using the Olympus OV-110 epifluorescenceimager. Very low green autofluorescence was detected inRosa26CreER^(T2);mT/mG mice when treated with vehicle control andirradiated with 365 nm UV, and strong EGFP signal was detected whentreated with 4-OHC (FIG. 13). In a second experiment,Rosa26CreER^(T2);mT/mG mice were injected intraperitoneally with caged4-OHC and subjected to UV illumination only on the ventral (but notdorsal) skin. Resultant EGFP signal was observed on the ventral skin butnot on the dorsal skin (FIG. 14) with an average 2.5-fold increase influorescence intensity. Because the increase of the EGFP signal wasconcomitant with the decrease of the tdTomato signal, EGFP/tdTomatoratios were also calculated in FIG. 14 for both ventral and dorsal skin.

A set of additional experiments was performed to determine whether caged4-OHC could be used to induce CreER activity in the mammary glands ofthe female Rosa26CreER^(T2);mT/mG mice following light exposure. Themice were injected with vehicle or caged 4-OHC, and only the rightmammary gland was exposed to the 365 nm light, whereas the left was not(FIG. 15). Seven days later, the right and left mammary glands wereresected for ex vivo imaging at a high spatial resolution using theOlympus Intravital Laser Scanning Microscope IV-110. The data show that365 nm UV illumination of the mammary gland alone did not causenoticeable green autofluorescence increase or tissue morphologicalchanges (FIG. 16). However, when caged 4-OHC was injected, the right,but not the left, mammary gland showed strong EGFP induction (FIG. 17).Taken together, the skin and mammary gland data clearly establish thatthe caged 4-OHC displayed superior in vivo inducibility by light andlimited diffusion after uncaging to affect other organs.

An additional set of experiments were performed to test the ability ofCaged Molecules I, II, and III (shown in Schematic #5) to induce nucleartranslocation of a ER fusion protein in a cell following light exposure.Mouse embryonic fibroblasts from Rosa26-CreER^(T2); Lox-STOP-Lox-lacZembryos were treated with 5 μM 4-OHC (positive control; lower leftpanel), treat Caged Molecule I (“Compound I”) with or without 365 nm UVlight exposure (top right and top center panels, respectively), CagedMolecule II (“Compound II”) with or without 365 nm UV light exposure(middle right and middle center panels, respectively), or Caged MoleculeIII (“Compound III”) with or without 365 UV light exposure (bottom rightand bottom left panels, respectively). The data show that irradiation ofCaged Molecules I, II, and III resulted in an increase in the nucleartransport of the ER fusion protein (a CreER fusion protein) in the cells(FIG. 18).

In sum, the data show that the presently provided caged tamoxifenderivative molecules can efficiently regulate CreER-mediatedrecombination in a light-dependent manner in mice. One major advantageof using caged tamoxifen and caged tamoxifen derivative molecules overother methods of making Cre activity photo-regulatable is that one canseamlessly integrate the light into numerous existing CreER models toachieve an additional level of stringent control, i.e., regional- andcell-specific control of gene expression. For example, thevillin-CreER^(T2) mouse allows efficient target gene recombinationthroughout the entire digestive epithelium in response to systemictamoxifen treatment (El Marjou et al., Genesis 39:186-193, 2004), and byrestricting light activation of caged tamoxifen or caged tamoxifenderivative molecules to the colon, this mouse strain may become anexcellent driver for spatiotemporal modeling of colorectal cancer inmice. Moreover, the application of this optochemogenetic (OCG) switch isnot confined to CreER; as described further herein, it can providephotoregulation of a spectrum of ER-fusion proteins. These ER fusionproteins would be subject to tamoxifen- or tamoxifenderivative-dependent control of nuclear localization and proteinactivity in the nucleus. For example, such a fusion protein can be atelomerase-ER fusion protein (Jaskelioff et al., Nature 469:102-106,2011). The OCG switch approach can use used in broad applications,especially in tumor and developmental biology, where localized andpattern-specific gene manipulation is of central importance to addressmany outstanding questions.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A composition, comprising:

or a pharmaceutically acceptable salt thereof.
 2. The composition ofclaim 1, wherein the composition comprises

or a pharmaceutically acceptable salt thereof.
 3. The composition ofclaim 2, wherein the composition is formulated as a liquid.
 4. A kitcomprising: one or more doses of the composition of claim 1; and a lightsource that emits light of a wavelength of between 350 nm to 410 nm. 5.A method of inducing nuclear translocation of a fusion proteincomprising a human estrogen receptor ligand binding domain in aeukaryotic cell, the method comprising: providing a eukaryotic cell thatcontains a fusion protein comprising a human estrogen receptor ligandbinding domain, contacting the eukaryotic cell with the composition ofclaim 1; and irradiating the eukaryotic cell with a wavelength of lightbetween 350 nm to 410 nm for a period of time sufficient to release4-hydroxycyclofen from the composition, wherein the released4-hydroxycyclofen induces the nuclear translocation of the fusionprotein.
 6. The method of claim 5, wherein the eukaryotic cell is in amammal.
 7. The method of claim 6, wherein the eukaryotic cell is presentin the mammary gland or the skin.
 8. The method of claim 6, wherein thecomposition is locally administered to a target tissue in the mammalthat contains the eukaryotic cell.
 9. The method of claim 5, wherein theeukaryotic cell comprises a nucleic acid encoding the fusion protein,and the nucleic acid is stably integrated into a chromosome of the cell.10. The method of claim 9, wherein the nucleic acid encoding the fusionprotein is: operably linked to a tissue-specific promoter sequence; oroperably linked to an inducible promoter sequence, and the eukaryoticcell is further contacted with a chemical inducing agent.
 11. The methodof claim 5, wherein the fusion protein comprises: a sequence of arecombinase, and the fusion protein has recombinase enzymatic activity;a sequence of a transcription factor, and the fusion protein is capableof promoting gene transcription in the nucleus of the eukaryotic cells;a sequence of a transcription repressor, and the fusion protein iscapable of repressing transcription of a gene in the nucleus of theeukaryotic cell; a sequence of a histone deacetylase and the fusionprotein has histone deacteylase activity; a sequence of a histoneacetyltransferase and the fusion protein has histone deacetylaseactivity; a sequence of an O-6-methylguanosine-DNA methyltransferase andthe fusion protein O-6-methylguanine-DNA methyltransferase activity; asequence of a telomerase, and the fusion protein has telomeraseactivity; or a sequence of an oncogene.
 12. The method of claim 5,wherein the eukaryotic cell is an undifferentiated cell, and the fusionprotein comprises a sequence of a transcription factor or transcriptionrepressor that induces cellular differentiation.
 13. A method ofinducing recombination in a eukaryotic cell, the method comprising:providing a eukaryotic cell that comprises (i) a nucleic acid encoding afusion protein comprising a sequence of a recombinase and a sequence ofa human estrogen receptor ligand binding domain, wherein the fusionprotein has recombinase activity, and (ii) a recombinase recognitionsequence that is specifically recognized by the fusion protein, whereinboth the nucleic acid encoding the fusion protein and the recombinaserecognition sequence are integrated into a chromosome within the nucleusof the eukaryotic cell; contacting the eukaryotic cell with acomposition of claim 1; and irradiating the eukaryotic cell with awavelength of light between 350 nm to 410 nm for a period of timesufficient to release 4-hydroxycyclofen from the composition, whereinthe 4-hydroxycyclofen stimulates the nuclear importation of the fusionprotein and the fusion protein stimulates recombination at therecombinase recognition sequence.
 14. The method of claim 13, whereinthe eukaryotic cell is in a mammal.
 15. The method of claim 14, whereinthe eukaryotic cell is present in the mammary gland or the skin.
 16. Themethod of claim 14, wherein the composition is locally administered to atarget tissue in the mammal that contains the eukaryotic cell.
 17. Themethod of claim 13, wherein the recombination results: in a decrease inthe expression of a transgene located between two recombinaserecognition sequences in the chromosome; in the replacement of asequence between two recombinase recognition sequences with a newtransgenic sequence; or in the increase in the proximity of a promoteror enhancer sequence to a transgene, wherein the recombination resultsin increased expression of the transgene.
 18. The method of claim 13,wherein the nucleic acid encoding the fusion protein is: operably linkedto tissue specific promoter sequence in the chromosome of the eukaryoticcell; or operably linked to an inducible promoter in the chromosome ofthe eukaryotic cell, and the eukaryotic cell is further contacted with achemical inducing agent.
 19. A method of treating breast cancer in asubject diagnosed as having breast cancer, the method comprising:administering to the subject an amount of the composition of claim 1sufficient to treat breast cancer in a subject, and irradiating themammary tissue of the subject with light between 350 nm and 410 nm,wherein the irradiating mediates the release of 4-hydroxytamoxifen inthe irradiated mammary tissue.
 20. The method of claim 19, wherein thecomposition is locally administered to the mammary tissue of thesubject.